Methods for patterning hydrogels into multi-well plates

ABSTRACT

The inventive subject matter provides methods for reproducibly fabricating hydrogel-based organ and tumor models inside multi-well plates. A hydrogel precursor, which can include cells, is instilled into a well. A pillar is inserted into the well to contact the hydrogel precursor with a surface that can be shaped or textured to provide a desired surface configuration or contour, for example that of a desired organoid or tumor feature. The hydrogel precursor is polymerized and the pillar removed. A second hydrogel precursor, which can contain a different cell type, is then instilled into the well and a second pillar, which can have a different configuration or texture, inserted. Subsequent polymerization generates a second hydrogel portion within the well. Polymerization can be carried out by photopolymerization. Different wells can be aligned with different, individually controlled light sources or a single, collimated light source.

This application is a continuation of U.S. patent application Ser. No.15/645,979, filed Jul. 17, 2017, and claims priority to U.S. provisionalpatent application No. 62/360,178, filed Jul. 8, 2016, and U.S.provisional patent application No. 62/411,438, filed Oct. 21, 2016. Allextrinsic materials identified herein are incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The field of the invention is apparatuses for fabricating engineeredtissues, namely tumor models, cardiac tissues, skin tissues, kidneytissues, neural tissues, liver tissues, cartilage tissues, and bonetissues for diagnostic and therapeutic screening.

BACKGROUND

The following background discussion includes information that may beuseful in understanding the present invention. It is not an admissionthat any of the information provided herein is prior art or relevant tothe presently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

In preclinical testing and clinical diagnostics, in vitro cellularassays are often used to test compound efficacy, toxicity, and host ofother measurable outcomes related the in vivo pathological condition.For high-throughput lead compound validation experiments in preclinicalscreening, 96-well, 384-well, or 1536-well microtiter plates areemployed to study cellular-compound interaction. Often these studies usea flat, glass or plastic (e.g., polystyrene) substrates to which cellsadhere and grow in a monolayer-called two-dimensional (2D) cell culture.Such 2D cell culture techniques have demonstrated inconsistencies withthe actual in vivo outcome, prompting the fields of engineering,biology, and medicine collectively to develop novel techniques to studycells at a more complex, three-dimensional (3D) physiologically relevantenvironment, called 3D cell culture. See e.g., W. Asghar, R. El Assal,H. Shafiee, S. Pitteri, R. Paulmurugan, U. Demirci, Engineering cancermicroenvironments for in vitro 3D tumor models, Materials Today, 2015,18 (10): 539-553; E. Knight, S. Przy Borski, Advances in 3D Cell CultureTechnologies enabling tissue-like structures to be created in vitro,Journal of Anatomy, 2015, 227 (6): 746-756.

Some progress has been made using scaffold-free and scaffold-basedapproaches to 3D cell culture. In scaffold-free methodologies, cellsaggregate into aggregates, which may have specific 3D shapes andplacement. For example, multicellular sphere-like aggregates, orspheroids, form using hanging-drop and magnetic levitation methods.Spheroids also form in cultures grown in containers, such asround-bottom wells, V- and U-shaped wells on microtiter plates, andultra-low attachment surfaces. See e.g., W. Asghar et al., supra;PCT/US2015/050522; U.S. Pat. No. 9,267,103B2; US20120171744A1;US20140322806A1.

However, scaffold-free techniques suffer from numerous disadvantages.First, scaffold-free techniques limit the user to studying spheroidgrowth and viability/toxicity in bulk measurements. Second,scaffold-free techniques do not take into account important aspects oftumor progression such as invasion, metastasis, and angiogenesis in thecontext of the tumor microenvironment (e.g., stromal cells, vascularcells, macrophages, extracellular matrix).

Scaffold-based techniques build 3D multi-cellular structures for 3Dassays from extracellular matrixes, biomaterials, and/or polymericstructures. In the resulting structures, cells collectively respond tomatrix, paracrine, and cellular cues to induce invasion, matrixdegradation, or differentiation. Scaffold materials include natural,animal-based materials (e.g., basement membrane extract, or Matrigel;collagen), plant-based materials (e.g., alginate), synthetic (e.g.,poly-ethylene glycol), or natural-synthetic combination and derivativematerials (e.g., methacrylated hyaluronic acid or methacrylic gelatin).See e.g., W. Asghar et al., supra; S. Caliari, J. A. Burdick, Apractical guide to hydrogels for cell culture, Nature Methods, 2016, 13(5): 405-414; E. Jabbari, “Three dimensional matrix for cancer stemcells”, patent application Ser. No. 14/527,028, October 2014.

Synthetic and synthetic-natural hybrid polymeric biomaterials offerconsiderable advantages in modular material properties, as well ascontrol over growth factor and binding site presentation andconcentration. Modified dextran (e.g., dextran methacylate), gelatin(e.g., gelatin methacrylamide and/or gelatin methacrylate), hyaluronicacid (e.g., hyaluronic acid methacrylate), polyvinyl alcohol (e.g.,acrylic acid modified PVA, acrylamide modified PVA) and polyethyleneglycol (e.g., polyethylene glycol diacrylate), their derivatives, andcombinations, have proven particularly useful in understanding diseaseprogression in 3D cellular models. See e.g., K. Nguyen, J. L. West,Photopolymerizable hydrogels for tissue engineering applications,Biomaterials, 2002, 23: 4307-4314; S. Pedron, A. C. Harley, Impact ofthe biophysical features of a 3D gelatin microenvironment onglioblastoma malignancy, Journal of Biomedical Research A, 2013,101(12): 3404-3415; B. Ananthanarayanan, Y. Kim, S. Kumar, Elucidatingthe mechanobiology of malignant brain tumors using a brainmatrix-mimetic hyaluronic acid hydrogel platform, Biomaterials, 2011,32: 7913-7923. However, there remains a need for apparatuses and methodsthat quickly and reproducibly fabricate such materials intotissue-mimetic cell culture scaffolds. In other words, apparatuses andprocesses that form cell culture scaffolds that have properties similarto the in vivo cellular environment by patterning structures (e.g.,venous structures) and controlling stiffness, density, porosity,adhesion properties, water content, pH, composition, heat and masstransport etc.

Recently, researchers have used photo-crosslinkable biomaterials forstudying cancer progression. Typically, a light-sensitive prepolymersolution is formulated and mixed with cells and polymerized using UVlight. A pattern within the matrix may be introduced via a photomaskplaced between the light source and the material, such that only thelight passing through the mask design will crosslink the light-sensitivematrix below. Typically, cancer cells are mixed with the prepolymersolution so that they can be encapsulated within the matrix. See e.g.,N. Peela, F. S. Sam, W. Christenson, D. Truong, A. W. Watson, G.Mouneimme, R. Ros, M. Nikkah, A three dimensional micropatterned tumormodel for breast cancer cell migration studies, Biomaterials, 2016, 81:72-83. Although this approach is simple, the resulting matrices fail toprovide an environment in which cells migrate as if they were in livingtissue, Rather, the individual cells may either adhere to one another,to the matrix, or escape the matrix and migrate out of the gel randomly.This random cell response leads to lower reproducibility and complicatesmeasurements of overall tumor growth and invasion.

A second hurdle in current 3D cancer cell culture is enabling long timepoints for tumor spheroid growth and invasion. Hanging drop studies arepractical for only tumor growth and viability studies, without anyability to study tumor cell invasion into extracellular matrix. Spheroidinvasion assays are typically performed in Matrigel. However, due toMatrigel's soft (<1 kPa) and easily cell degradable matrix, experimentsmay not last beyond several days. Cellular processes such as invasion,angiogenesis, and transdifferentation (whereby tumor cells differentiateinto other cell types) may be take weeks. One research groupincorporated a tumor spheroid into a polymerized matrix that was wedgedbetween two molds. However, invasion and quantifiable growth were notobserved, which may be due to the experimental setup and lack ofnutrient transport to the tumor. See e.g., A. Aung, J. Theprungsirikul,H. L. Lim, S. Varghese, Chemotaxis-driven assembly of endothelialbarrier in a tumor-on-a-chip platform, Lab on a chip, 2016, 16:1886-1898. Thus, a platform having improved durability and mass (e.g.,nutrient, oxygen, carbon dioxide, waste etc.) transport is needed forstudying and assaying controlled invasion and tumor growth over longtime points.

A third hurdle for cellular assay analysis is studying 3D tumor cellculture in a high-throughput manner (e.g., multi-well plates) withoutthe possibility of culture disruption during liquid media changes(removal via aspiration and re-application via pipetting) and for easyimaging. Currently in both spheroid-only and gel-based assays, sampleloss or disruption to the microenvironment is likely to occur,diminishing reproducibility. It is highly necessary to limit anyexternal (e.g., human) errors during media exchange in 3D cell culture.

To address the technical challenges posed by these hurdles, hydrogelshave been patterned by extrusion bio-printing into each well, but againusing a well-by-well process. These processes are laborious in naturewhen having to scale into multi-well plates for higher-throughputassays. Cell viability issues arise when cells remain in pre-polymersolution for long durations. And an extrusion process that uses a finetip causes shear stress that can damage or kill cells, and is limited tousing only soft, shearing materials, which are not representative of thetumor microenvironment. It would be beneficial to pattern gels into allwells at once, with the flexibility of material stiffness, to enhancescalability, reproducibility, and customization of the hydrogelstructures and increase viability of the cells within and on the gels.

Some progress has been made by forming hydrogels using digital lightprojection (DLP), stereolithography (SLA), or general photo-patterning,between a glass slide and coverslip, and the resulting gel on glass isadded to each well of a multi-well plate. In DLP, the projected image,or mask, is limited by the projector optics, which can cause imageclarity and light uniformity issues across the area of the mask.

For example, WO 2015/179572 to Chung et al. discloses a system for 3Dmicrofabrication that projects light toward a light modulator thatmodulates light responsive to digital masks corresponding to layers of astructure. A series of images corresponding to the digital masks areprojected on a photopolymerizable material while a stage controls theoptical plane. However, the system requires sophisticated equipment, andfabrication in multi-well plates can only be done using multiple systemsor rapidly scanning across multiple wells.

Nikkhah et al. disclose a comparatively simple system in Patent Pub. No.US 2017/0067025. Nikkhah creates a high stiffness construct bycross-linking a first solution that includes cancer cells within aspacer. A second solution is disposed around the high stiffnessconstructs and crosslinked. Migration of cancer cells from the highstiffness construct to the low stiffness construct can then be observed.However, Nikkhah does not appreciate (1) that hydrogel-based tumormodels can be rapidly fabricated within multi-well plates in parallelusing a mold to control the height of the hydrogels and (2) that a spacebetween the sides of the hydrogels and the well walls facilitatesnutrient transport and exchange of liquid media. See also U.S. Pat. No.8,906,684 to Bhatia et al.

These and all other extrinsic materials discussed herein areincorporated by reference in their entirety. Where a definition or useof a term in an incorporated reference is inconsistent or contrary tothe definition of that term provided herein, the definition of that termprovided herein applies and the definition of that term in the referencedoes not apply.

It has yet to be appreciated that organ models (e.g., tumor modelscomprising tumor and non-tumor cells in hydrogels having stiffnessesthat mimic tumor and healthy tissue, respectively) may be easilyprepared. Thus, there is still a need for apparatuses and processes formaking organ, tissue, and tumor models that exhibit more accurate drugresponses, especially in high-throughput diagnostics and therapeuticassays.

SUMMARY OF THE INVENTION

The inventive subject matter provides methods for patterning multi-layerhydrogels into one or more wells of a multi-well plate viaphotopolymerization in combination with a pillar inserted into each ofthe wells. For example, such methods can be used to pattern tumor cellsin a hydrogel having a stiffness or pattern that corresponds to tumorsin vivo, or to generate organoids of controlled configuration within thewells of a plate.

One embodiment of the inventive concept is a method of forming ahydrogel in a well by adding a first prepolymer to the well, inserting afirst post (with a surface that contacts the first prepolymer onceinserted) into the well, and exposing the first prepolymer to aphotopolymerizing light to initiate polymerization of the firstprepolymer. A wash step can be performed (for example, using PBS orculture media) to remove excess first prepolymer from the well. Thesesteps can be repeated using a second prepolymer and a second post togenerate an additional polymer within the well, and can be followed byadding liquid media to the well. The posts can have different crosssections, and the contacting surface of the post can have differenttextures or patterns, for example corresponding to different features oforganoids or tumor models. The prepolymers used can include differentcell populations. For example, the first prepolymer can include tumorcells such as tumor cells obtained from primary culture, tumor cellsobtained from cell lines, cancer stem cells, spheroid, and/ormulti-cellular aggregate. In some embodiments cells of the secondprepolymer are nontumor cells, such as stromal cells, endothelial cells,pericytes, organ-specific cells t-type, cancer associated fibroblasts,lymphocytes, macrophages, endothelial cells, dendritic cells, bonemarrow derived cells, mesenchymal stem cells, endothelial progenitorcells, normal epithelial cells, and/or granulocytes. In some embodimentsthe cells of the first prepolymer and/or the second prepolymer can becardiomyocytes, cardiac fibroblasts, endothelial cells, vascular smoothmuscle cells, parietal cells, podocytes, mesangial cells, keratinocytes,fibroblasts, and melanocytes, hepatocytes, fibroblasts, endothelialcells, and Kupffer cells, and/or stem cells.

The light used for photopolymerization steps can pass through differentpassageways. For example, light used for the first photopolymerizationstep can pass through a first light passageway and light used for asecond photopolymerization step can pass through a second lightpassageway. These different passageways can provide illumination todifferent portions of the well. In some embodiments the above steps canbe carried out on more than one well of a plate. In such embodimentseach well can be illuminated by individual, controlled light sources,which are controlled to provide even illumination between the differentwells. In other embodiments a single, collimated light source can beused.

Preferably, tumor models produced using the inventive apparatuses adhereto the bottom of each well of the multi-well plate. A space between sidesurface(s) of the tumor model and the walls of each well allow forliquid media to contact both top and side surfaces of the tumor model.The ability to exchange the media with minimal hydrogel disruption moreclosely replicates the in vivo tumor environment and increases thelength of time over which the tumor model can be cultured. Thegeneration of such tumor models can be useful in studying metastasis inpatient tumors and known cell lines, and to test therapeutic agents in ahigh throughput manner.

Apparatuses according to the inventive subject matter comprise a lightsource, a photomask, a holder that aligns the photomask and themulti-well plate, and a mold that configures surfaces of hydrogels inthe various wells of the multi-well plate.

Suitable light sources emit light having energy (wavelength(s)) andpower that initiates polymerization and/or crosslinking of prepolymer(e.g., monomers/macromers). Although use of a single, diffuse lightsource (e.g., a mercury or other light bulb) is contemplated, preferredembodiments comprise an array of light-emitting diodes (“LEDs”). EachLED of the LED array is positioned to illuminate one well of amulti-well plate. For example, when a multi-well plate is stacked on topof a holder, photomask, and light source, each LED of the LED array iscentered under one well of the a multi-well plate. Advantageously, LED'semit less heat than incandescent bulbs, decreasing the likelihood thatthe heat will damage or kill the cells in the pre-polymer mixture. Itshould be appreciated that the light source emits light in the UV and/orvisible ranges to initiate hydrogel polymerization using photoinitiatorsthat have excitation wavelengths in the UV and/or visible ranges,respectively. For example, a light source that has peak emissionwavelengths of 300-500 nm, 350-450 nm, and preferably about 365 or 405nm are suitable for use in the inventive apparatus. The emissionwavelength can be further tuned using filters and/or monochromators.

The light source can be powered by either battery or electricity and canbe controlled using simple switches, timers, or programmed controllers(e.g., an Arduino® controller). In an exemplary embodiment of theinventive subject matter, a controller controls light intensity,wavelength, on-time, and/or other lighting parameters. When an LED arrayis used, the controller can individually control such parameters foreach LED. Optionally, the controller can be configured to automaticallyturn the light source on when the mold is inserted in the multi-wellplate.

Contemplated multi-well plates include those with which researchers arealready familiar and those that can be used in existing imagingequipment, for example MatTek™ glass bottom plates having, e.g., 35 mm,50 mm, 60 mm, or 100 mm dishes having microwell diameters of 7 m, 10 mm,14 mm, 20 mm, or 30 mm. The inventors further contemplate that theinventive apparatuses could be adapted for use with the ThermoScientific™ Nunc™ Lab-Tek™ II Chamber Slide™ System (having 1, 2, 4, or8 wells per slide system), Greiner Sensoplates, and/or Matsunami glassbottom dishes.

Photomasks allow light to pass through the pre-polymer to initiatepolymerization. Contemplated photomasks have opaque regions to blocklight and light passageways (e.g., holes or transparent regions) toallow light to illuminate the multi-well plate and prepolymer solution.For example, one light passageway allows light to illuminate a centralportion of the bottom of a well while excluding an edge region.Conversely, another light passageway allows light to illuminate an edgeregion of the bottom of a well, but excludes the central portion. Thephotomask can also include light passageways that allow light toilluminate the bottom of each well, including the edges. This wouldresult in hydrogels that extend up the sides of the posts and the wallsof each well. Such hydrogels can also be obtained when the apparatus isused without a photomask. Alternatively, light passageways can bepartially opaque (translucent, but not transparent) in some areas tooffer further flexibility in resulting polymerization, gelationproperties, and gel structures.

In one exemplary embodiment, a photomask having a diameter less than theinternal diameter of the wells is used to create a tumor cell-containinghydrogel. Then, a second photomask having a light passage with a largerdiameter is used to polymerize a non-tumor cell-containing outer layerthat contacts the top and sides of the tumor cell-containing hydrogel.In another exemplary embodiment of the inventive subject matter, thesame photomask is used in the first and second photopolymerization stepsto form a second hydrogel layer having the same diameter as the firsthydrogel layer on the first hydrogel layer, without fully encasing thesides of the first layer.

The size of each light passageway can also be varied. Contemplated lightpassageways can be configured to transmit light to no more than 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% etc. of the cross-sectional areaof the bottom of one or more wells. Light passageways can have a varietyof shapes, including, but not limited to: lines, circles, ovals,triangles, squares, pentagons, hexagons, dot arrays, venous structures,etc.

Holders align the photomasks and multi-wells plate above the lightsource. To fabricate multilayer tumor models, one photomask is used topattern each layer. Therefore, the holder allows for successivephotomasks to be positioned under the multi-well plate during each stepof the tumor model fabrication process. Holders may have a variety ofsuitable configurations. For example, a holder may comprise a recess,slot, drawer, etc. configured to retain the photomask. To alignmulti-well plates with the photomask, a holder also includes a recess,slot, or other mechanism for retaining multi-well plates.

Molds control the thickness and the contours of the top surface of eachlayer of the tumor models. Molds have posts that dip into prepolymersolution dispensed into each well of the multi-well plate. The diameterof each post allows enough space between each post and the walls of eachwell to allow the prepolymer mixture displaced by the post to rise ineach well. To control the thickness of each layer, the bottom end ofeach post is positioned at a distance from the bottom of a well andmolds the top surface of the resulting polymer structure. Employingmolds improves consistency of hydrogel height and makes the top layer ofeach polymer flat. Whereas without molds, the top surface of each layermay curve with the meniscus of the prepolymer mixture. Theseadvantageous effects are achieved, because the posts come in contactwith the prepolymer solution resulting in a planar polymerization planeand reach through the oxygenated zone of the prepolymer mixture wherepolymerization would be quenched. The mold may also reflect light,enhancing polymerization.

Molds can be disposable or reusable (sterilizable). The number of postscan be selected to match the number of wells in the multi-well plate.The size and shape of each post can also be varied. For example, theposts can have different lengths and/or different horizontalcross-sections.

In one embodiment of the inventive subject matter, the apparatus isassembled by simply stacking each element. Another embodiment includes ahousing configured to orient light from the light source toward themulti-well plate. Optionally, the housing may also be configured tocontrol hydrogel thickness by controlling the distance between thebottom of mold posts and the bottom of the wells. For example, thehousing can include rails, tension rods, slots, adjustable pegs, a setof height adapters (e.g., inserts having different thicknesses), a screwjack, a servo, an actuator, or other structure configured to lower/raisethe mold such that the post(s) dip into the well(s) to set distance(s)from the bottom of the well(s).

Yet another embodiment of the inventive subject matter includes a secondholder that retains the mold and adjusts relative positions of the moldand the plate. Such holders can also be configured to removably retainmolds, so that different molds can be used in the apparatus including:disposable or reusable molds, molds with differing numbers of posts foruse with multi-well plates having different numbers of wells, and/ordifferent post designs.

An enclosure can be used to limit operator's exposure to UV light. Forexample, the enclosure could be disposed around at least a portion ofthe multi-well plate to shield the operator from light leakage.Contemplated enclosures are black or transparent. Transparent enclosuresabsorb/filter harmful wavelengths of light but allow the operator toview the process.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an exploded view of an apparatus and a 24-well plate.

FIG. 1B shows a photomask nested in a holder.

FIG. 1C shows another embodiment of an apparatus according to theinventive subject matter and a 24-well plate.

FIGS. 2A-D shows perspective, side, and bottom views of a mold for a24-well plate.

FIG. 3A shows a schematic representation of photopolymerization usingthe inventive apparatus.

FIG. 3B shows a close-up view of one well of the scheme shown in FIG.3A.

FIG. 3C shows a hydrogel in liquid media in the well shown in FIG. 3B.

FIGS. 4A-C show exemplary photomask designs.

FIGS. 5A-B show exemplary light sources.

DETAILED DESCRIPTION

The inventors have devised an apparatus for preparing a high-throughput3D cellular assay for studying tissue growth, viability, and highthroughput drug screening. For example, tumor assays for studyinggrowth, viability, and invasion and migration through an extracellularmatrix that is representative of the tumor microenvironment in terms ofcomposition and stiffness. One should appreciate that the disclosedapparatus and techniques provide many advantageous technical effectsincluding the ability to reproducibly tune the composition and stiffnessof in vitro hydrogels to mimic the extracellular matrix microenvironmentof tumors in vivo. Additionally, the planar hydrogel surfaces formedusing the inventive apparatus enable superior real-time brightfield andfluorescence imaging.

The following discussion provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

FIG. 1A shows an exploded view of the elements one embodiment of theinventive subject matter. The apparatus includes mold 120, multi-wellplate 130, photomask 140, light source 150, and holder 160. Themulti-well plate shown is a standard 24-well plate, so the photomask 140has 24 light passages, and light source 150 has 24 LEDs. FIG. 1B showsphotomask 140 nested in holder 160. When the elements are stacked, theholder aligns the light passages of photomask 140 and each LED of lightsource 150 with the wells of the 24-well plate.

In addition to 24-well plates, the inventor contemplates thatapparatuses can be modular in design such that various elements aredesigned to work with different multi-well plates (e.g., 6-well,12-well, 24-well, 48-well, 96-well, 384-well, 1536-well plates). Forexample, the number of posts in mold 120, the number of light passagesin photomask 140, and the number of LEDs in light source 150 typicallymatch the number of wells in the multi-well plate. In addition tomulti-well plates apparatus designs can be configured for use withMatTek glass bottom dishes, Thermo Scientific™ Nunc™ Lab-Tek™ II ChamberSlide™ Systems, and/or Matsunami glass bottom dishes.

The apparatus shown in FIG. 1C includes housing 110, enclosure 155, andtop enclosure 115. Holder 160 includes a slot for inserting photomask140. Advantageously, different photomasks can easily be inserted/removedfrom the slot in holder 160 to form different hydrogel layers. Ratherthan a slot, holder 160 can include a drawer for removably retainingdifferent photomasks.

FIGS. 2A-D show various views of mold 220 and posts 221. FIG. 2A is aperspective view. FIG. 2B shows a bottom view of mold 220, looking downthe axis of posts 221. FIGS. 2C and 2D show side views of mold 220having 6 and 4 posts 221, respectively. Suitable molds do notsubstantially adhere to the hydrogel after polymerization. For example,molds made of polydimethylsiloxane (“PDMS”) or Teflon® release thehydrogels after polymerization without damaging them. Alternatively,molds can comprise a nonstick coating, (e.g., PDMS or Teflon®). Theapparatus may be used without the mold. However, molds improve thereproducibility of hydrogel fabrication by molding the surface andcontrolling the height of the hydrogels.

Although the molds shown have posts with smooth, planar bottom surfaces,the bottom of each post can also be textured and/or patterned to molddifferent organoid/tissue structures. For example, the bottom of eachpost can comprise channel molds to yield hydrogels having channels thatmimic vascular structures. See e.g., WO2016141137A1 to Lewis et al. Inanother example, the bottom of each post can comprise a renal cortexmold that forms an organoid model of the renal cortex afterpolymerization of one layer, then the medulla can be polymerized in thenext layer or vice versa. The inventive apparatus can also be employedto simultaneously fabricate and seed tissue engineered organs such asthe colon, esophagus, and pancreas models as described in U.S. patentapplication Ser. No. 10/150,828 to Grikscheit et al. In yet anotherexemplary embodiment of the inventive subject matter, the mold cancomprise a mold for honey comb structures to form liver tissues asdescribed by Shepherd et al. in US20160272946A1.

A schematic representation of fabricating a hydrogel using the inventiveapparatus is shown in FIGS. 3A-C. Each well 331 of multi-well plate 330holds prepolymer 381. Mold 320 is placed on top of multi-well plate 330.Posts 321 dip into prepolymer 381 in each well 331. Light from lightsource 350 passes through light passages 341 of photomask 340,initiating polymerization of prepolymer 381. Polymerization zone 382 isshown in close-up FIG. 3B. After the polymerization step is complete,excess prepolymer 381 is washed away (e.g., by aspiration using PBS),and liquid media 390 is added to well 331 and contacts the sides and topsurface of hydrogel 383. Although a single hydrogel layer is shown,additional polymerization steps can be performed to add additionalhydrogel layers.

Exemplary hydrogel prepolymers comprise dextran, collagen, gelatin,hyaluronic acid, polyvinyl alcohol, and/or polyethylene glycol. Theprepolymer and the resulting hydrogel can also comprise an extracellularmatrix, an extracellular matrix component, and/or an extracellularmatrix derivative. One having ordinary skill in the art would appreciatethat the extracellular matrix component may include laminin,fibronectin, elastin, and/or fibrin. The prepolymer solution can alsocomprise cell binding and signaling peptides and proteins. The peptideand/or protein component can include one or more of Streptavidin,biotinylated-proteins, acrylated-RGD peptide, VEGF protein, and so on.See e.g., Gjorevski N. et al. Designer matrices for intestinal stem celland organoid culture. Nature 2016, 539, 560-564.

The greater the degree of crosslinking between polymer chains, thegreater the hydrogel stiffness. The inventors contemplate that thestiffness of a tumor cell-containing hydrogel can range from 1 Pascals(Pa) to 100,000 kiloPascals (kPa), including the end points. For tumormodels, the stiffness of the tumor cell-containing hydrogel is between10 Pa and 100 kPa, and even more preferably between 1 kPa and 40,000kPa, inclusive. Bone and cartilage models have stiffnesses that mayexceed 100 kPa. See e.g., WO2016142323A1 to Schaufelberger et al. Unlessthe context dictates the contrary, all ranges set forth herein should beinterpreted as being inclusive of their endpoints, and open-ended rangesshould be interpreted to include commercially practical values.Similarly, all lists of values should be considered as inclusive ofintermediate values unless the context indicates the contrary.

In further regard to hydrogel stiffness, any suitable means ofcrosslinking is contemplated, for example, chemical or physicalcrosslinking. Preferably, chemical crosslinking is achieved byphotocrosslinking. The inventors contemplate that suitablephotoinitiators are UV or visible light-responsive, soluble, andminimally cytotoxic before and after polymerization. As used herein theterm minimally cytotoxic means that the photoinitiator kills no morethan 50%, and preferably no more than 10%, of the tumoral or nontumoralcells disposed in the prepolymers used to prepare the first and secondmatrices as measured after polymerization. For example any Irgacure®photoinitiator may be suitable.

In this schematic, the thickness of the resulting hydrogel, 383 (FIG.3C) is determined by the length of each post 321. The thickness ofhydrogel 383 can be controlled using molds having posts with differentlengths, using spacers, holders, etc.

FIG. 4A shows a schematic representation of photomask 440 with circularlight passages 441 having a variety of diameters. For example, thehydrogel pattern can comprise a plurality of circles each having a widthof 25-5000 microns and a height of 25-2000 microns. Photomasks can bemade of opaque plastic or metal with cutout areas that serve as lightpassages. Photomasks can also be made from transparency film (e.g., withsilver halide ink) or soda lime/quartz (with chrome). A photomask mayalso comprise light passageways, which comprise a plurality of irisesthat can be adjusted to different diameters.

FIG. 4B is a schematic representation of photomask 440 having lightpassages 441 having different shapes and sizes. Although circles,triangles, squares, and hexagons are shown, the photomask can comprise aplurality of polygons (e.g., squares, rectangles, pentagons, hexagons,heptagons, octagons, etc.), wherein each polygon of the plurality ofpolygons has a width of 50-5000 microns and a height of 50-2000 microns.

FIG. 4C shows a photomask having a plurality of linear light passages.Exemplary photomasks yield hydrogel patterns that comprise a pluralityof lines each having a width of 25-5000 microns and a height of 25-2000microns.

FIG. 5A shows light source 550 having 96 LEDs 551, and FIG. 5B showslight source 550 having 384 LEDs 551. It should be appreciated that thenumber of LEDs need not necessarily match the number of wells in themulti-well plate used in the apparatus. For example, a 96 or 184 LEDarray may provide sufficiently uniform light to produce uniformhydrogels in a 24-well array. Conversely, 24 LEDs can be configured toreproducibly produce uniform hydrogels in multi-well arrays having morethan 24 wells by varying the direction of the light emitted, thedistance from the light source, and the photomask design. Optionally, aheat skink may be included to limit temperature changes and damage tothe LEDs, which is preferable for some prepolymer solutions.

The apparatus can further comprise a plurality (e.g., a bundle or anarray) of optical fibers (and any necessary couplers/optical equipment).In some embodiments, hydrogel patterning within the multi-well plate (orother container) can be achieved by illuminating the prepolymer viaindividually (or selectively) addressable optical fibers, without aphotomask. Advantageously, the light source can be positioned remotefrom the cell laden prepolymer, decreasing the risk that heat from thelight source will accelerate polymerization or damage the cells.

In yet another embodiment, the light source and mold are configured toilluminate the prepolymer from above. For example, the mold can haveposts made of a light conducting material, such as poly(methylmethacrylate) (e.g., Lucite), quartz, or glass to deliver light to theprepolymer in addition to controlling hydrogel height/thickness. Anopaque coating on the mold can be used to control the pattern of lightemitted from the posts. For example, the coating may cover the portionof the mold from which each post extends as well as the side surfaces ofeach post, leaving uncoated portions for light entry and exit from themold. The bottom of each post can further be patterned with lines,polygons, or other shapes in the manner described above in regard tophotomasks.

To prepare tumor models using apparatuses according to the inventivesubject matter, a first hydrogel is patterned in each well of amulti-well plate using photolithography, wherein the first hydrogel hasa first stiffness and encapsulates a tumor cell. A second hydrogel ispatterned in each well of the multi-well plate using photolithographyand at least partially contacts the first hydrogel. The second hydrogelhas a second stiffness, different from the first stiffness andencapsulates a non-tumor cell. The inventive subject matter furtherincludes tumor models that optionally feature a third hydrogel and athird cell type either encapsulated in the third hydrogel or disposedbetween the second and third hydrogels.

With respect to the tumor cells, contemplated tumor cells include tumorcells from primary culture, tumor cells from cell lines, and/or cancerstem cells. In preferred embodiments, the tumor cell further comprises aplurality of tumor cells, which form a spheroid.

Suitable nontumoral cells include a stromal cell, an endothelial cell, apericyte, and/or an organ-specific cell t-type. Therefore, the nontumorcell can comprise one or more of cancer associated fibroblasts,lymphocytes (T cells, B cells, and/or natural killer cells),macrophages, endothelial cells, dendritic cells, bone marrow derivedcells, mesenchymal stem cells, endothelial progenitor cells, normalepithelial cells, and granulocytes (eosinophils, basophils, and/orneutrophils).

To model cardiac tissues, the cells may comprise cardiomyocytes, cardiacfibroblasts, endothelial cells, and/or vascular smooth muscle cells,etc. Kidney models can comprise parietal cells, podocytes, and/ormesangial cells. Skin models can comprise keratinocytes, fibroblasts,and melanocytes. Liver models can comprise hepatocytes, fibroblasts,endothelial cells, and Kupffer cells. See e.g., US20160272946A1 toShepherd et al. Neurons and glial cells can be employed in brain tissuemodels. In any tissue model, stem cells (adult, embryonic, inducedpluripotent, or other stem cells) can be included to further mimic thetarget cellular microenvronment and/or to regenerate tissue-specificcells.

The cells may be labeled with fluorescent dyes or proteins (e.g.,green-fluorescent protein, red-fluorescent protein, CellTracker™,mCherry, and so on) to distinguish individual cell populations and totrack tumor growth and invasion in the context of the microenvironmentcells.

The inventive subject matter offers several advantages: spatialpatterning of chemically (and photochemically) crosslinkable materialswith a broad range of material properties (e.g., stiffness andcomposition) enables a more realistic extracellular matrix for tumor andnon-tumor compartments of a tumor assay to match physiologicalconditions in vivo; sequential patterning of multiple cell types on thetumor microenvironment (e.g., tumor vs. non-tumor cells) enables morecontrolled evolution of cell interactions; precise placement of tumorand non-tumor cell types in 3D enables observation of tumor progressioninto non-disease tissue, and allows dissection of various components ofthe microenvironment, including a changing ECM, the role of various celltypes, and tumor response to various drug treatments; the evolving tumorgrowth and invasion through the matrix and onto the planar matrixsurface enables image clarity and quantifiable measurements of growth,viability and invasion in response to drug treatment; 3D gel constructsfabricated in a well of a tissue culture dish, glass slide, ormulti-well plate where there is a space between the gel and well wallenables efficient liquid media exchange during aspiration and pipettingand minimal disruption to the gel.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

As used herein, and unless the context dictates otherwise, the term“coupled to” is intended to include both direct coupling (in which twoelements that are coupled to each other contact each other) and indirectcoupling (in which at least one additional element is located betweenthe two elements). Therefore, the terms “coupled to” and “coupled with”are used synonymously.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A method of forming a hydrogel in a first well,comprising; dispensing a first prepolymer into the first well; insertinga first post into the first well, wherein the first post comprises afirst contact surface that contacts the first prepolymer upon insertion;exposing the first prepolymer to a first illumination, therebyinitiating polymerization of the first prepolymer; performing a washstep to remove excess first prepolymer; dispensing a second prepolymerinto the first well; inserting a second post into the first well,wherein the second post comprises a second contact surface that contactsthe second prepolymer upon insertion; exposing the second prepolymer toa second illumination, thereby initiating polymerization of the secondprepolymer; and dispensing a liquid media into the first well.
 2. Themethod of claim 1, wherein the first contact surface and the secondcontact surface have different cross sections.
 3. The method of claim 1,wherein the wash step comprises aspiration with phosphate bufferedsaline (PBS) or cell culture media.
 4. The method of claim 1, whereinthe first contact surface comprises a first texture or pattern.
 5. Themethod of claim 4, wherein the first texture or pattern is configured tomold a first organoid or a first tissue structure.
 6. The method ofclaim 1, wherein the second contact surface comprises a second textureor pattern.
 7. The method of claim 6, wherein the second texture orpattern is configured to mold a second organoid or a second tissuestructure.
 8. The method of claim 1, wherein the first prepolymercomprises a plurality of first cells and the second prepolymer comprisesa plurality of second cells.
 9. The method of claim 8, wherein theplurality of first cells are tumor cells.
 10. The method of claim 9,wherein the tumor cells are selected from the group consisting of tumorcells obtained from primary culture, tumor cells obtained from celllines, and cancer stem cells.
 11. The method of claim 10, wherein theplurality of tumor cells comprise a spheroid, or multi-cellularaggregate.
 12. The method of claim 8, wherein the plurality of secondcells are nontumor cells.
 13. The method of claim 12, wherein thenontumor cells are selected from the group consisting of stromal cells,endothelial cells, pericytes, organ-specific cells t-type, cancerassociated fibroblasts, lymphocytes, macrophages, endothelial cells,dendritic cells, bone marrow derived cells, mesenchymal stem cells,endothelial progenitor cells, normal epithelial cells, and granulocytes.14. The method of claim 8, wherein at least one of the plurality offirst cells and the plurality of second cells are selected from thegroup consisting of cardiomyocytes, cardiac fibroblasts, endothelialcells, vascular smooth muscle cells, parietal cells, podocytes,mesangial cells, keratinocytes, fibroblasts, melanocytes, hepatocytes,fibroblasts, endothelial cells, and Kupffer cells, and a stem cell. 15.The method of claim 1, wherein the first post and the second post areconfigured to form a tissue engineered organ or a tissue structure, andwherein the first prepolymer comprises a plurality of third cells andthe second prepolymer comprises a plurality of fourth cells.
 16. Themethod of claim 15, wherein at least one of the plurality of third cellsand the plurality of fourth cells are selected from the group consistingof cardiomyocytes, cardiac fibroblasts, endothelial cells, vascularsmooth muscle cells, parietal cells, podocytes, mesangial cells,keratinocytes, fibroblasts, melanocytes, hepatocytes, fibroblasts,endothelial cells, neurons, glial cells, and Kupffer cells, and a stemcell.
 17. The method of claim 1, wherein the first illumination occursthrough a first light passageway and the second illumination occursthrough a second light passageway.
 18. The method claim 17, wherein thefirst light passageway provides illumination of a first area of thefirst well and the second light passageway provides illumination of asecond area of the first well.
 19. The method of claim 1, comprising theadditional steps of: dispensing a third prepolymer into a second well ofa fixture comprising the first well; inserting a third post into thesecond well, wherein the third post comprises a third contact surfacethat contacts the first prepolymer upon insertion; and exposing thethird prepolymer to a third illumination during the first illumination,thereby initiating polymerization of the third prepolymer.